Innamorati Giulio, Piccirillo Rosanna, Bagnato Paola, Palmisano Ilaria, Schiaffino Maria Vittoria
DIBIT, Scientific Institute San Raffalele, Via Olgettina 58, 20132 Milan, Italy.
Pigment Cell Res. 2006 Apr;19(2):125-35. doi: 10.1111/j.1600-0749.2006.00292.x.
The protein product of the ocular albinism type 1 gene, named OA1, is a pigment cell-specific integral membrane glycoprotein, localized to melanosomes and lysosomes and possibly implicated in melanosome biogenesis. Although its function remains unknown, we previously showed that OA1 shares structural similarities with G protein-coupled receptors (GPCRs). To ascertain the molecular function of OA1 and in particular its nature as a GPCR, we adopted a heterologous expression strategy commonly exploited to demonstrate GPCR-mediated signaling in mammalian cells. Here we show that when expressed in COS7 cells OA1 displays a considerable and spontaneous capacity to activate heterotrimeric G proteins and the associated signaling cascade. In contrast, OA1 mutants carrying either a missense mutation or a small deletion in the third cytosolic loop lack this ability. Furthermore, OA1 is phosphorylated and interacts with arrestins, well-established multifunctional adaptors of conformationally active GPCRs. In fact, OA1 colocalizes and coprecipitates with arrestins, which downregulate the signaling of OA1 by specifically reducing its expression levels. These findings indicate that heterologously expressed OA1 exhibits two fundamental properties of GPCRs, being capable to activate heterotrimeric G proteins and to functionally associate with arrestins, and provide proof of principle that OA1 can actually function as a canonical GPCR in mammalian cells.
1型眼白化病基因的蛋白质产物,名为OA1,是一种色素细胞特异性整合膜糖蛋白,定位于黑素小体和溶酶体,可能参与黑素小体的生物发生。尽管其功能尚不清楚,但我们之前表明OA1与G蛋白偶联受体(GPCR)具有结构相似性。为了确定OA1的分子功能,特别是其作为GPCR的性质,我们采用了一种常用于在哺乳动物细胞中证明GPCR介导信号传导的异源表达策略。在这里我们表明,当在COS7细胞中表达时,OA1表现出相当大的自发激活异源三聚体G蛋白和相关信号级联的能力。相比之下,在第三个胞质环中携带错义突变或小缺失的OA1突变体缺乏这种能力。此外,OA1被磷酸化并与抑制蛋白相互作用,抑制蛋白是构象活性GPCR的成熟多功能衔接蛋白。事实上,OA1与抑制蛋白共定位并共沉淀,抑制蛋白通过特异性降低其表达水平来下调OA1的信号传导。这些发现表明,异源表达的OA1表现出GPCR的两个基本特性,即能够激活异源三聚体G蛋白并在功能上与抑制蛋白相关联,并提供了OA1在哺乳动物细胞中实际上可以作为典型GPCR发挥作用的原理证明。